1. Field of the Invention
The present invention relates to a pipette adapter suitably usable for measuring the absorbance of a sample in the field of pharmaceutical industry and the like, an absorbance measuring pipette comprising this pipette adapter and a pipette, and a tip attachable to this pipette adapter, as well as an absorbance measuring apparatus and absorbance measuring method for measuring the absorbance of a sample by using this absorbance measuring pipette.
2. Related Background Art
In a wide range of fields such as pharmaceutical industry, food industry, chemical industry, agriculture, forestry, and fishery, samples are analyzed by measuring their absorbance for studying and developing new drugs, screening enzymes, analyzing microorganisms, and so forth. Among such analyzing methods, examples of methods for analyzing biological samples, such as nucleic acids, proteins, and the like, which are important in bio-related fields, include the following:
(1) Since biological samples as a specimen exist by only a minute amount in general, and their quantitative determination is also important, a sample is transferred to a specific cell for measuring a minute amount. The cell containing this sample is irradiated with test light. The intensity of test light transmitted through the cell and sample is detected, and the absorbance of the sample is measured according to the result of this detection. In this case, for putting the sample into the cell, a pipette 10 and a tip 30, such as those as shown in FIG. 11, are used. The tip 30 is detachably attachable to the front end of the pipette 10, and the sample is metered into the tip 30.
(2) U.S. Pat. No. 5,844,686 discloses a method using a tip having a window for introducing test light or a reflecting mirror for reflecting the test light, attaching this tip to the front end of a pipette, and measuring the absorbance of a sample while the latter is contained in the tip. This method aims at improving the recovery ratio of the sample, preventing the sample from being contaminated upon recovery, and carrying out rapid measurement of absorbance.
The inventors have found that the following problems exist in the above-mentioned conventional absorbance measuring methods. Namely:
1) In samples such as nucleic acids, proteins, and the like, subsequent reactions are often carried out after the measurement of absorbance. In such a case, it is necessary for the sample contained in the cell for absorbance measurement to be recovered after the measurement. However, the method of above-mentioned (1) is problematic in that the recovery ratio of the sample is insufficient, the sample is contaminated upon recovery, the cell is hard to wash, and so forth.
2) The method of the above-mentioned (2), on the other hand, has problems as follows. In general, for preventing the problem of contamination from occurring, the tip is used only once and is discarded without being reused. Providing such a disposable tip with the above-mentioned window or reflecting mirror is inappropriate in that the tip becomes expensive. For reusing the tip without discarding it, washing is necessary for the tip also acting as a cell. Further, since the tip has a window for introducing test light or a reflecting mirror for reflecting the test light, it is difficult for the tip to be made smaller, whereby the invention disclosed in the above-mentioned publication is not effective for a sample having a small amount (minute amount in particular).
Therefore, in order to overcome the above-mentioned problems, it is an object of the present invention to provide a pipette adapter, an absorbance measuring pipette, an absorbance measuring apparatus, and an absorbance measuring method, by which the step of recovering a sample can be omitted, the sample can be prevented from being contaminated upon recovery, no specific cell for measurement is necessary, and the absorbance of a small amount of sample can be measured by use of an inexpensive tip.
For achieving such an object, the inventors have accomplished the present invention. Namely, the pipette adapter in accordance with the present invention is one usable together with a pipette for measuring an absorbance of a sample including a specimen, attachable between the pipette and a tip adapted to contain the sample, having an inner space continuing to respective internal spaces of the pipette and tip when attached thereto, and comprising test light introducing means for introducing test light into the inner space from outside and emitting the test light toward a sample suction port of the tip.
Thus configured pipette adapter is used as being attached between the pipette and the tip. In its attached state, the respective internal spaces of the pipette adapter, pipette, and tip are continuous. By the test light introducing means, the test light is introduced into the inner space of the pipette adapter from outside and is reflected toward the sample suction port of the tip. As a consequence, the test light is transmitted through the sample contained in the tip, whereby the absorbance of the sample can be measured. As the pipette and tip, those conventionally sold and utilized can be employed. Also, a tip made of an inorganic material such as glass, stainless steel, or the like can be used.
Preferably, the test light introducing means of the pipette adapter in accordance with the present invention comprises a test light introducing window for introducing the test light into the inner space from outside and a reflecting mirror for reflecting toward the sample suction port of the tip the test light introduced into the inner space through the test light introducing window. In this case, the test light is introduced into the inner space of the pipette adapter through the test light introducing window from outside and is reflected by the reflecting mirror so as to be emitted toward the sample suction port of the tip.
Preferably, the test light introducing means comprises an optical fiber which emits, from one end thereof disposed within the inner space toward the sample suction port of the tip, the test light guided therethrough from outside. As a consequence, the test light is guided through the optical fiber from outside and is emitted toward the sample suction port of the tip from one end of the optical fiber located within the inner space of the pipette adapter. More preferably, in this case, test light collecting means, disposed near the above-mentioned one end of the optical fiber, for collecting the test light is further provided.
Also, more preferably, the test light introducing means selectively emits toward the sample suction port of the tip only a predetermined wavelength band component of the test light introduced into the inner space from outside. As a consequence, wavelength components unnecessary for absorbance measurement can be restrained from irradiating the sample, whereby the sample within the tip can be prevented from raising its temperature.
The absorbance measuring pipette in accordance with the present invention comprises the pipette adapter in accordance with the present invention and a pipette attachable to the pipette adapter. The pipette adapter and the pipette may be detachable from each other or may be used as being integral with each other. In the case where they are detachable from each other, the pipette adapter can be washed easily as necessary. When they are integral with each other, on the other hand, their handling can be improved.
It will be useful if a tip having an inserting portion, in a substantially conical or pyramidal form, for receiving the pipette adapter, and a sample container portion, in a tubular form, having an end part formed with the sample suction port is further provided. If such a tip is used for drawing in the sample by suction, and the sample is held in the sample container portion of the tip, then the reproducibility in measuring the absorbance of the sample can be enhanced. If the tubular sample container portion is made narrower and longer, then the transmission length of the test light can be enhanced even when the amount of the sample is minute.
The absorbance measuring apparatus in accordance with the present invention is an apparatus for measuring an absorbance of a sample including a specimen and comprises (1) a light source for outputting test light; (2) the absorbance measuring pipette in accordance with the present invention introducing the test light outputted from the light source into an inner space, attaching thereto a tip adapted to contain the sample, and reflecting the test light toward the sample suction port of the tip; and (3) a detection optical system for detecting the test light outputted outside from the sample suction port of the tip attached to the absorbance measuring pipette.
In thus configured absorbance measuring apparatus, the test light outputted from the light source is introduced into the inner space of the absorbance measuring pipette, reflected toward the sample suction port of the tip attached to the absorbance measuring pipette, and outputted to the outside from the sample suction port of the tip, thereby being detected by the detection optical system. By use of the result of this detection, the absorbance of the sample in the tip is measured.
Specifically, it is preferred that arithmetic means for computing the absorbance of the sample in the tip according to an intensity of the test light detected by the detection optical system in a state where the sample is contained in the tip and an intensity of the test light detected by the detection optical system in a state where the sample is not contained in the tip or in a state where a blank sample including no specimen is contained in the tip is further provided.
If the detection optical system is capable of simultaneously or substantially simultaneously detecting intensities of a plurality of components having wavelengths different from each other in the test light outputted outside from the sample suction port of the tip, then the respective absorbance values of the sample in a plurality of wavelength components can be measured substantially simultaneously.
In addition, it is preferred that the absorbance measuring apparatus in accordance with the present invention further comprise temperature adjusting means for cooling at least the tip or causing the tip to keep a constant temperature. As a consequence, the volumetric change of air within the tip, such as thermal expansion in particular, caused by the rise or change in temperature of the tip or its surroundings is suppressed. In this case, it will be further preferable if the surroundings of the tip are also cooled.
Preferably, the pipette adapter has a side wall at least a part of which is in a conical or pyramidal form, and holding means having a bored portion to which a predetermined part of the side wall of the pipette adapter in a conical or pyramidal form is adapted to fit is further provided. As a consequence, the absorbance measuring pipette can be attached and detached quite easily. Also, the absorbance measuring pipette is held stably and firmly, whereby the misalignment of optical axis in the optical path of the test light can be reduced.
The absorbance measuring method in accordance with the present invention is a method of measuring an absorbance of a sample including aspecimen, which is a method favorably carrying out absorbance measurement for the sample by using the absorbance measuring pipette in accordance with the present invention. Namely, the absorbance measuring method in accordance with the present invention comprises a step of attaching a tip adapted to contain the sample to the absorbance measuring pipette of the present invention; a step of letting the tip to contain the sample or a blank sample including no specimen; a step of introducing test light into the inner space of the absorbance measuring pipette from outside and detecting the test light outputted to the outside from the sample suction port of the tip; and a step of calculating the absorbance of the sample contained in the tip according to an intensity of the test light detected in a state where the sample is contained in the tip and an intensity of the test light detected in a state where the sample is not contained in the tip or in a state where the blank sample is contained in the tip.
In the step of detecting the test light, it is preferred that intensities of a plurality of components having wavelengths different from each other in the test light outputted to the outside from the sample suction port of the tip be detected simultaneously or substantially simultaneously. Also, in the step of detecting the test light, it is preferred that the test light be detected while at least the tip is cooled or while the temperature of the tip is held constant.
The tip in accordance with the present invention is attachable to the pipette adapter of the present invention; and has a sample container portion containing a sample including a specimen, the sample container portion having a tubular form (shaped like either a cylinder or a polygonal tube) and having substantially parallel inner walls in a cross section taken along a center axis. Such a tip can enhance the reproducibility of optical path length of the test light passing through the sample. Further, the present invention provides a tip which is attachable to the pipette adapter of the present invention and is formed from a light-shielding member capable of substantially blocking test light irradiating a sample including a specimen. If such a tip is used for measuring the absorbance of a sample, there is substantially no possibility that part of the test light irradiating the sample may pass through the tip and enter a photodetector.